The immune response to specific antigens is regulated by the recognition of peptide fragments of those antigens by T lymphocytes. Within an antigen presenting cell (APC), peptide fragments of a proteolytically processed antigen become bound into the antigenic peptide binding site of major histocompatibility complex (MHC) molecules. These peptide-MHC complexes are then transported to the cell surface for recognition (of both the foreign peptide and the adjacent surface of the presenting MHC molecule) by T cell receptors on helper or cytotoxic T lymphocytes. That antigen-specific recognition event initiates the immune response cascade for either protective or deleterious immune responses.
Two classes of MHC molecules function as immune system presenters of antigenic peptides to T cells. MHC class I molecules receive peptides from endogenously synthesized proteins, such as an infectious virus, in the endoplasmic reticulum about the time of synthesis of the MHC class I molecules. The MHC class I-bound antigenic peptides are presented at the cell surface to CD8-positive cytotoxic T lymphocytes, which then become activated and can kill the virus-expressing cells directly. In contrast, MHC class II molecules are synthesized in the endoplasmic reticulum with their antigenic peptide binding sites blocked by the invariant chain protein (Ii). These MHC class II-Ii protein complexes are transported from the endoplasmic reticulum to a post-Golgi compartment where Ii is released by proteolysis and a specific antigenic peptide becomes bound to the MHC class II molecule.
The Ii protein is cleaved by intracellular proteases through a series of fragments, some of which remain associated with the MHC class II molecules. This series of fragments has been better defined through the treatment of cultured, [35S]methionine-labeled cells with certain protease inhibitors. For example, leupeptin and antipain block the action of respective classes of proteases on Ii, and on Ii fragments which remain associated with the MHC class II alpha and beta chains. The MHC class II-bound fragments of Ii are recognized after immunoprecipitations with anti-MHC class II antibodies and/or anti-Ii antibodies, gel electrophoresis and autoradiography. In vitro cleavages of immunopurified MHC class II alpha, beta-Ii protein complexes with cathepsin B, cathepsin D, and other proteases, define site specific cleavages by individual enzymes. The MHC class II alpha, beta chains are relatively resistant to proteolysis.
These specific cleavage sites in Ii have been confirmed at a molecular level with Ii mutants having amino acid replacements at putative sites for proteolysis. Several cleavage sites were defined. The crucial site for understanding the mechanism of the compounds of this invention is in a region of clustered cationic-hydrophobic dipeptidyl units in human Ii (77-92) (Lu et al, J. Biol. Chem. 145: 899-904, (1990)). Mutation at each of these four, redundant cleavage sites in the mutant Ii [R78xe2x86x92A; K80xe2x86x92A; K83xe2x86x92A; K86xe2x86x92T] blocks cleavage in that region (Xu et al., Molecular Immunology 31: 723-731 (1994)).
The region with these clustered, apparent cleavage sites lies in the primary sequence of Ii about the positions of N-termini of a series of naturally occurring Ii fragments, the CLIP peptides. The CLIP peptides occur naturally in isolated MHC class II molecules and are abundantly presented in MHC class II molecules of a mutant cell line which is deficient in some mechanism which regulates antigenic peptide charging into MHC class II molecules. This last finding has led to the hypotheses that the CLIP peptides are an intermediate in peptide charging into MHC class II molecules (Roche, P., and Cresswell, P., Nature 345: 615-619 (1990)), or represent a default pathway to block such molecules from accepting ambient peptides after charging with an APC-selected peptide has failed (Xu et al. in Antigen Processing and Presentation, Humphreys, R. E., ed.: 228-242, Academic Press, NY (1994)).
Overlap among the MHC class II molecule binding sites for antigenic peptide, the Ii-CLIP peptides, and the therapeutic Ii-key peptide, is being determined by x-ray crystallography at a molecular level. The exact position of influenza virus hemagglutinin peptide HA307-319 in the antigenic peptide binding groove HLA-DR1 was determined first (Stern et al., Nature 368: 215-221 (1994)). Subsequently, the exact positioning of a CLIP peptide in the same antigenic peptide binding groove was determined (Ghosh et al., Nature 378: 457-462 (1995)). In both cases, the peptides assumed the conformation of a polyprolyl type II helix in the antigenic peptide binding groove. The backbone atoms of the CLIP peptide overlay exactly the positions of the backbone atoms of the HA peptide, with comparable placement of side chains into pockets of the MHC class II molecule. Residue position M91 of the CLIP peptide overlays the first residue position of the HA peptide. The CLIP residues N-terminal to M91, extending back to P87 were also in a polyprolyl type II helix conformation. More N-terminal residues, including positions human Ii L77-K83 were not resolved in those crystallographic studies, but clearly lie outside the antigenic peptide binding groove, along the side of the MHC class II molecule.
Thus, although much has been learned with respect to the interaction of molecules in the antigen presentation process, the application of relevant findings to therapeutic ends remains, for the most part, unrealized.
The present invention relates, in one aspect, to a class of compounds referred to herein as effector compounds. Effector compounds are useful in connection with the modulation of an immune response. Modulation refers to the ability of the effector compounds of the present invention to either enhance (antigen supercharging), or inhibit (immunosuppressant activities) antigen presentation, depending upon the nature of the particular effector compound, and the therapeutic context.
Effector compounds include peptides and modified peptides. In a preferred embodiment, the invention relates to the mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) and modifications thereof, the peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) being specifically excluded. Modifications specifically demonstrated include, for example, the deletion of amino acids from the N-terminus; the deletion of amino acids from the C-terminus; the protection of the C-terminus; the protection of the N-terminus; N-terminal extensions; substitutions; and cyclized derivatives. The invention also encompasses peptidomimetic structures which are structurally and functionally related to the effector compounds listed above.
Thus, the present invention relates to methods for enhancing presentation of an MHC class II restricted antigenic peptide to a T cell. Such methods include contacting the following components under physiological conditions: an MHC class II expressing antigen presenting cell; the mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) or modifications thereof (the peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) being specifically excluded); the MHC class II restricted antigenic peptide which, when added to the incubation mixture, is not in association with an antigen presenting cell; and a T cell which is responsive to the MHC class II restricted antigenic peptide.
In another aspect, the present invention relates to methods for inhibiting presentation of an MHC class II restricted antigenic peptide to a T cell. Such methods include contacting the following components under physiological conditions and incubating for an appropriate period: an MHC class II expressing antigen presenting cell displaying on its surface a T cell-presented epitope from a native protein antigen; and mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) and modifications thereof (the peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) being specifically excluded).
In other embodiments, the invention relates to effector compounds (i.e., peptides, modified peptides or peptidomimetics) which induces release of an antigenic peptide specifically from a human MHC class II allele in the absence of another antigenic peptide which binds to the human MHC class II allele. A preferred embodiment is the peptide YRMKLPKSAKPVSQMR (SEQ ID NO:3), or deletion modifications wherein from 0 to 4 amino acid residues are deleted from the C-terminus and from 0 to 6 amino acid residues are deleted from the N-terminus.
The invention also relates to an effector compound which induces release of a first antigenic peptide from a human MHC class II allele in the presence of a second antigenic peptide which binds to the human MHC class II allele. Other specific embodiments include effector compounds which bind allosterically to modulate antigenic peptide binding into the antigenic peptide binding site of human MHC class II molecules; allele-specific modulators of antigen presentation; and locus-specific modulators of antigen presentation.
The present invention also relates to a second class of compounds, referred to herein as immunomodulatory organic compounds. Such compounds are identified by a method which includes the following steps: providing a first complex comprising an MHC class II molecule to which an antigenic peptide has been bound; contacting the first complex with mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) (or modifications thereof including peptidomimetics), thereby forming a second complex; and screening organic molecules for compounds which bind to the second complex but not to the first complex, and which exhibit immunomodulatory activity. Compounds identified in this manner can be used to modulate an immune response in a mammal.